The invention relates to novel RGS (regulators of G-protein signaling) nucleic acids and proteins. Also provided are vectors, host cells, and recombinant methods for making and using the novel molecules.
Regulators of G-protein signaling (RGS) accelerate guanosine triphosphate (GTP) hydrolysis by Gi, but not by Gs class xcex1-subunits (Popov et al. (1997) Proc. Natl. Acad. Sci. USA 94:7216-20). RGS proteins were first identified in genetic screens in fungi and nematodes as negative regulators of G-protein signaling (Dolhman et al. (1995) Mol. Cell. Biol. 15:3635-43). RGS proteins have been shown to function as GTPase-activating proteins. It has additionally been proposed that RGS proteins may act as effector antagonists by occluding the effector-binding sites on G-protein xcex1-subunits (Helper et al. (1997) Proc. Natl. Acad. Sci. USA 94:428-432).
RGS has been implicated in a distinct molecular mechanism with the potential to modulate G-protein responses. Proteins containing the RGS domain can directly control aspects of G-protein stimulated signaling pathways. RGS proteins appear to enhance the endogenous GTPase activity of G-proteins, thus decreasing the half-life of the active GTP-bound state and limiting the duration of Gxcex1i signaling.
The glucose-dependent insulinotropic peptide receptor (GIP-R) is a member of the G-protein coupled receptors. GIP was first isolated from porcine small intestine and was described as a member of the secretin family of gastrointestinal regulatory peptides (Tseng and Zhang (1998) Endocrin. 139:4470-75). In the presence of glucose, GIP is a potent stimulator of insulin release by pancreatic islet xcex2-cells. GIP may represent an important hormonal mediator in the entero-insular axis. Insulinotropic properties of GIP in diabetic patients have been shown to be diminished despite elevated serum levels of GIP. While the precise mechanism for the decline in insulinotropic activity of GIP in diabetic patients has not been defined, agonist-induced desensitization of G-protein-coupled receptors is well documented (Premont et al. (1995) FASEB J. 9:175-182).
Recently, an interaction of the G-protein with members of RGS proteins has been demonstrated to mediate a desensitization mechanism. RGS proteins act as GTPase activating proteins to decrease the half-life of the activated G xcex1-subunit (Koelle et al. (1996) Cell 84:115-125; Druey et al. (1996) Nature 379:742-46).
Additionally, RGS proteins may be involved in cell migration. Cell migration is a required behavior in the development and maintenance of multicellular organisms. Generally, cells migrate in response to various chemoattractants and chemorepellents in the environment (Bowman et al. (1998) J. Biol. Chem. 273:28040-48). Chemoattractants provide a directional signal to cells leading to migration of the cells towards the source of the chemoattractant (Butcher et al. (1996) Science 272:60-66; Mackay, C. R. (1996) J. Exp. Med. 184:799-802). Chemoattractants also direct the rapid, integrin-dependent adhesion of leukocytes to various cell-associated or extracellular proteins if the corresponding chemoattractant receptor is expressed at high levels. RGS proteins appear to be involved as most leukocyte chemoattractants mediate their activity by binding and stimulating specific Gxcex1-coupled receptors.
RGS proteins constitute a family of proteins characterized by an RGS domain. A number of RGS proteins have been identified and several have been shown to function as GTPase-activating proteins (Chatterjce et al. (1997) Genomics 45:429-33). Identification of other members of the RGS family are needed.
Because of the complexity of the immune response and regulation of heterotrimeric G-protein signaling, additional mechanisms are needed to modulate such functions. Additionally, methods are needed to regulate an immune response, and provide therapies for a range of diseases.
Isolated nucleic acid molecules corresponding to regulators of G-protein signaling (RGS) nucleic acid sequences are provided. Additionally amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NOS:2 and 4 or the nucleotide sequences encoding the DNA sequence deposited in a bacterial host as ATCC Accession Number 207048, or the DNA sequence obtained from the overlapping clones deposited as ATCC Accession Numbers 207049 and 207050. By xe2x80x9cDNA sequence obtained from the overlapping clonesxe2x80x9d is intended that the DNA sequence of the human sequence can be obtained by sequencing of the two individual clones which together comprise the entire human sequence. Further provided are RGS polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein.
The present invention also provides vectors and host cells for recombinant expression of the nucleic acid molecules described herein, as well as methods of making such vectors and host cells and for using them for production of the polypeptides or peptides of the invention by recombinant techniques.
The RGS molecules of the present invention are useful for modulating the phenotype of immune and respiratory responses, particularly for regulating an immune response. The molecules are useful for the diagnosis and treatment of immune and respiratory disorders, including, but not limited to, atopic conditions, such as asthma and allergy, including allergic rhinitis, psoriasis, the effects of pathogen infection, chronic inflammatory diseases, organ-specific autoimmunity, graft rejection, and graft versus host disease. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding RGS proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of RGS-encoding nucleic acids.
Another aspect of this invention features isolated or recombinant RGS proteins and polypeptides. Preferred RGS proteins and polypeptides possess at least one biological activity possessed by naturally occurring RGS proteins.
Variant nucleic acid molecules and polypeptides substantially homologous to the nucleotide and amino acid sequences set forth in the sequence listings are encompassed by the present invention. Additionally, fragments and substantially homologous fragments of the nucleotide and amino acid sequences are provided.
Antibodies and antibody fragments that selectively bind the RGS polypeptides and fragments are provided. Such antibodies are useful in detecting the RGS polypeptides as well as in regulating G-protein signaling.
In another aspect, the present invention provides a method for detecting the presence of RGS activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of RGS activity such that the presence of RGS activity is detected in the biological sample.
In yet another aspect, the invention provides a method for modulating RGS activity comprising contacting a cell with an agent that modulates (inhibits or stimulates) RGS activity or expression such that RGS activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to RGS protein. In another embodiment, the agent modulates expression of RGS protein by modulating transcription of an RGS gene, splicing of an RGS mRNA, or translation of an RGS mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the RGS mRNA or the RGS gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant RGS protein activity or nucleic acid expression by administering an agent that is an RGS modulator to the subject. In one embodiment, the RGS modulator is an RGS protein. In another embodiment, the RGS modulator is an RGS nucleic acid molecule. In other embodiments, the RGS modulator is a peptide, peptidomimetic, or other small molecule.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of the following: (1) aberrant modification or mutation of a gene encoding an RGS protein; (2) misregulation of a gene encoding an RGS protein; and (3) aberrant post-translational modification of an RGS protein, wherein a wild-type form of the gene encodes a protein with an RGS activity.
In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of an RGS protein. In general, such methods entail measuring a biological activity of an RGS protein in the presence and absence of a test compound and identifying those compounds that alter the activity of the RGS protein.
The invention also features methods for identifying a compound that modulates the expression of RGS genes by measuring the expression of the RGS sequences in the presence and absence of the compound.
Other features and advantages of the invention will be apparent from the following detailed description and claims.